Avian immunoglobulin-producing cell lines

Abstract

The present invention involves a method for preparing antibody-producing chicken cell clones. This method comprises a series of steps including initially immunizing a first chicken with a desired antigen. A population of antibody-producing lymphocytes from bursa or spleen of the first chicken is separated. The antibody-producing lymphocyte population is then infected with helper-free reticuloendotheliosis virus REV-T or helper-free reticuloendotheliosis virus REV-T and CSV (preferably REV-T(CSV) and transplanted into a second chicken, the second chicken having been pretreated to remove normal B cells. The transplanted lymphocytes to proliferate in the second chicken, preferably for a period of at least about two weeks. The lymphocytes from spleen, bursa or peripheral blood of the second chicken are isolated and plated out, for example in microtiter plates. Cell clones producing antibody such as IgG or IgM directed against the desired antigen are then selected. The isolation step most preferably involves cell culturing in the microtiter plates for a period of at least about one week.

What is claimed is: 
     
       1. A method for preparing antibody-producing avian cell clones specific for a desired antigen, the method comprising: immunizing a bird with a desired antigen;   separating a population of antibody-producing B-lymphocytes from said bird;   treating said antibody-producing B-lymphocyte population in vitro with v-rel under conditions inducing transformation; and   incubating said v-rel-treated B-lymphocyte population in vitro under conditions facilitating proliferation of antibody-producing B-lymphocytes transformed by v-rel.   
     
     
       2. A method of preparing antibody-producing avian cell clones specific for a desired antigen, the method comprising: immunizing a bird with a desired antigen;   separating a population of antibody-producing B-lymphocytes from said bird;   treating said antibody-producing lymphocyte population in vitro with reticuloendotheliosis virus and a non-cytotoxic avian helper virus to induce cell transformation; and   incubating said treated B-lymphocyte population in vitro under conditions facilitating proliferation of transformed antibody-producing B-lymphocytes.   
     
     
       3. A method for preparing antibody-producing avian cell clones specific for a desired antigen, the method comprising: immunizing a first bird with a desired antigen;   separating a population of antibody-producing B-lymphocytes from the first bird;   treating said antibody-producing B-lymphocyte population in vitro with v-rel to produce transformed antibody-producing B-lymphocytes;   transplanting said treated B-lymphocytes into a second bird, the second bird having been pretreated to remove normal B-lymphocytes, and allowing transformed B-lymphocytes to proliferate in the second bird; and   isolating said transformed antibody-producing B-lymphocytes from the second bird.   
     
     
       4. The method of claim 1 or 2 wherein the incubating step comprises treatment of the treated B-lymphocyte population with the desired antigen and subsequent plating in microtiter plates. 
     
     
       5. The method of claim 1 or 2 wherein the incubating step comprises incubation of the treated B-lymphocyte population in culture medium comprising the desired antigen. 
     
     
       6. The method of claim 1 or 2 wherein the incubating step comprises incubation of the treated B-lymphocyte population in culture medium comprising B-cell mitogens. 
     
     
       7. The method of claim 6 wherein the B-cell mitogens are one or more of lectins, cytokines and antibodies directed against B-lymphocyte surface proteins. 
     
     
       8. The method of claims 1, 2, 3 wherein the separating involves panning said antibody-producing B-lymphocytes on solid surface comprising bound antibodies having binding specificity for avian IgM. 
     
     
       9. The method of claims 1, 2, 3 wherein the population of antibody-producing B-lymphocytes is separated from bursa, spleen, bone marrow, gland of Harder, intestinal lining or peripheral blood of the bird. 
     
     
       10. The method of claim 1 or 3 wherein the v-rel is included in a reticuloendotheliosis virus. 
     
     
       11. The method of claim 1, 2, 3 or wherein the bird is a chicken. 
     
     
       12. The method of claim 1 or 3 wherein the treating step involves transfection with a v-rel gene. 
     
     
       13. The method of claim 1 or 3 wherein the treating step involves electroporation. 
     
     
       14. The method of claim 1, 2, 3 or wherein the antibody produced is of an IgM isotype. 
     
     
       15. The method of claim 2 wherein the reticuloendotheliosis virus with a non-cytotoxic helper virus is REV-T(CSV). 
     
     
       16. The method of claim 2 wherein the helper virus is chick syncytial virus, spleen necrosis virus (SNV), attenuated REV-A or duck infectious anemia virus (DIAV). 
     
     
       17. The method of claim 1 or 2 wherein the incubating step involves plating cells out in microtiter plates. 
     
     
       18. The method of claim 1 or 2 wherein the incubating step involves cell culturing in microtiter plates for a period of at least about one week. 
     
     
       19. The method of claim 1 or 2 wherein the bird is between about six weeks and two years of age. 
     
     
       20. The method of claim 1, 2, 3 or wherein the immunization is monitored by routine serological analysis. 
     
     
       21. The method of claim 1, 2, 3 or wherein the immunization is indicated by measurement of an antibody titer greater than about 1/200. 
     
     
       22. The method of claim 1, 2, 3 or wherein the separation of antibody-producing B-lymphocytes involves centrifugation in a density gradient comprising polysaccharide. 
     
     
       23. The method of claim 1 or 2 wherein an isolating step involving plating out transformed B-lymphocytes, follows the incubating step. 
     
     
       24. The method of claim 1 or 2 wherein an isolating step involving selecting cell clones producing antibody directed against the desired antigen, follows the incubating step. 
     
     
       25. The method of claim 1 or 2 wherein an isolating step involving panning said transformed B-lymphocytes on a solid surface comprising a bound second antibody having a specific binding specificity for an avian antibody, follows the incubating step.